2 Current address: Pharmaceutical Research and Development,
Genentech, Inc., 460 Pt. San Bruno Blvd., South San Francisco, CA
94080, USA

3 Current address: Division of Pharmaceutics and
Pharmaceutical Chemistry, College of Pharmacy, The Ohio State
University, Columbus, OH 43210, USA

* To whom correspondence should be addressed.

H. R. Costantino

S. P. Schwendeman

R. Langer

A. M. Klibanov

Received July 7, 1997
The successful use of proteins in pharmaceutical and other commercial
applications requires close examination of their relative fragility.
Because of the resultant enhanced stability, proteins are often
formulated in the solid state, even though dehydration tends to alter
their structure. Even in the solid form, however, proteins may become
inactivated due to various deleterious processes, e.g., aggregation.
This review focuses on such mechanisms, with an emphasis on case
studies conducted in our laboratory. Proteins which have both disulfide
bonds and free thiols may aggregate via thiol-disulfide exchange, and
this process may be facilitated by lyophilization-induced structural
perturbations. For proteins possessing disulfides but not free thiols,
aggregation also may occur when native disulfides are
beta-eliminated, thus giving rise to thiol species which can
catalyze disulfide scrambling. Other deleterious processes have also
been uncovered, including a formaldehyde-mediated aggregation of
formalinized vaccines. It is illustrated how knowledge of such
deterioration pathways makes possible the rational development of
stable solid protein formulations.
KEY WORDS: lyophilized pharmaceutical proteins, stability

Proteins are finding ever-increasing use in therapeutic and other
commercial applications. And yet, proteins are relatively fragile
(compared to low-molecular-weight compounds) and prone to various
pathways of deterioration, as reviewed elsewhere [1-4]. This fragility adversely
impacts and potentially limits their development.

Therapeutic proteins are often formulated in the solid state, e.g.,
lyophilized, to yield a pharmaceutically acceptable shelf-life [5]. Furthermore, some novel controlled-delivery
systems for pharmaceutical proteins, such as sustained-release from
polymeric vehicles [6], also incorporate
lyophilized proteins. In addition, solid enzymes suspended in organic
solvents are valuable synthetic catalysts [7]. In
order to successfully employ solid proteins for all these purposes, it
is necessary to investigate and understand their deterioration in the
lyophilized state. This topic, despite its practical significance, is
foreign to classical biochemists who are used to dealing with proteins
dissolved in water.

Protein deterioration in the solid state may take the form of both
chemical and conformational. Griebenow and Klibanov [8] have quantified the (reversible) secondary
structural alteration of numerous proteins upon lyophilization. This
phenomenon may be relevant to chemical instability since in aqueous
solution a change in protein structure often leads to increased
susceptibility towards further deleterious processes, e.g., aggregation
[1-3]. On the other hand, there
have been reports that the alteration of protein structure upon
dehydration may actually improve stability upon lyophilization [9] or during storage in the dried state [10]. However, in some instances there is no
correlation between structural conservation and solid-state stability
[11, 12].

This review presents several instructive case studies using model
lyophilized proteins. We have focused on solid-state deterioration, in
particular protein aggregation. Our approach has been to elucidate the
mechanisms responsible for the deterioration of the protein in order to
facilitate the subsequent development of rational strategies for their
stabilization.

CASE STUDY OF ALBUMIN. AGGREGATION VIA THIOL-DISULFIDE
INTERCHANGE

In an early mechanistic study, Liu et al. [13]
investigated the solid-state aggregation of bovine serum albumin (BSA)
lyophilized from pH 7.3. Albumin is a pharmaceutically relevant
molecule [14, 15] often
chosen as a model for evaluation of sustained-release vehicles [16]. Following storage at elevated temperature
(37°C) and water content (~40 g/100 g dry), a loss of BSAs
solubility was observed upon reconstitution. This solubility loss was
attributed to protein aggregation occurring in the wetted solid.

The mechanism responsible for moisture-induced deterioration of BSA was
elucidated by dissolving the aggregates in specially designed aqueous
solutions. Since strong denaturants such as 6 M urea or guanidine
hydrochloride were incapable of solubilizing the aggregates, it was
concluded that the aggregates were covalently bonded. Furthermore, it
was shown that the covalent linkages were disulfides, since thiol
agents such as dithioerythritol (DTE) were effective in solubilizing
the aggregates. Aggregation via thiol-disulfide interchange is one of
several typical mechanisms of deterioration uncovered for lyophilized
proteins (see the table).

Some examples of deterioration of pharmaceutical proteins in the
lyophilized state

Subsequently, we examined the solid-state stability of recombinant human
albumin (rHA), which has a high sequence homology to BSA [17]. It was found [18] that rHA,
like its bovine counterpart, was susceptible to aggregation as a result
of storage at 37°C and high moisture (96% relative humidity, where
the water content was approximately 50 g/100 g dry protein). The time
course of aggregation of rHA under these conditions is depicted in Fig.
1a.

Fig. 1. Deterioration of some lyophilized pharmaceutical
proteins. Depicted are the time courses of the loss of solubility upon
reconstitution (as compared to an unincubated control) for rHA stored
at 37°C and 96% relative humidity (a), human insulin stored at
50°C and 96% relative humidity (b), and tetanus toxoid stored at
37°C and 84% relative humidity (c). All proteins were
excipient-free and lyophilized from pH 7.3.

The rHA aggregates were completely solubilized by DTE, indicating that
they were disulfide-linked. The albumin molecule (both BSA and rHA)
contains 17 cystine residues and one free thiol group of Cys-34 [17]. It was proposed and verified experimentally that
the solid-state aggregation of albumin was due to a thiol-disulfide
interchange [13, 18]. This
process is triggered by a nucleophilic attack by a thiol (in the form
of the thiolate ion) of one protein molecule on a sulfur atom of the
cystine disulfide of another. The result is a new, intermolecular
disulfide with conservation of the free thiolate ion. Since the process
is catalytic with respect to the latter species, subsequent additional
exchanges continue to take place, eventually yielding
high-molecular-weight insoluble aggregates.

It was also found that the solid-state aggregation of albumin was
dependent on the level of relative humidity, or more specifically, on
the water content of the wetted protein. For example, for rHA the
dependence of the extent of aggregation was bell-shaped with respect to
the water content, with maximal aggregation occurring at about 50 g/100
g dry protein [18]. A similar bell-shaped
dependency was also observed for the solid-state aggregation of BSA
which also undergoes the thiol-disulfide exchange [13].

To understand the effect of water on albumin aggregation, one has to
consider the fundamental role of water in the properties of the solid
protein. An increase in the water content leads to increased reactivity
for proteins due to several factors including [19]: 1) the ability of water to act as a
"molecular lubricant" (increasing the conformational
flexibility of proteins); 2) direct participation of water in
deleterious processes; and 3) the ability of water to facilitate the
diffusion of reactants. In the case of solid-state aggregation of
albumin, the ability of water to increase the conformation mobility of
the protein molecules should promote the intermolecular thiol-disulfide
reaction, thus explaining the ascending part of the aforementioned
bell-shaped curve.

The descending portion of the albumin aggregation versus water content
bell-shaped curve is most likely the result of a "dilution"
effect [13] and rearrangement of the protein
structure upon rehydration [18]. The latter
scenario was proposed for rHA, which undergoes a significant reversible
alteration of the secondary structure, as revealed by Fourier-transform
infrared (FTIR) spectroscopy [8, 20]. FTIR spectroscopy has proven to be a useful
technique for investigating the secondary structure of proteins in both
the aqueous and solid states [8, 9]. Figure 2 depicts FTIR spectra
for aqueous and lyophilized rHA in the amide III region. The spectra
show significant differences between the aqueous and dehydrated
samples, reflecting a difference in the protein secondary structure.
Gaussian curve-fitting analyses of the data indicate that upon
lyophilization the alpha-helix content of rHA dropped from 58%
in aqueous solution to 25-35% with a concomitant rise in both
beta-sheet and unordered structures. Thus, the rHA molecule is
less ordered in the dried state. Such a disordering is likely to
increase the accessibility of disulfides (which are otherwise buried in
the native albumin structure [21]). This scenario
is consistent with the observation that albumin aggregation via
thiol-disulfide exchange in aqueous milieu is greatly accelerated under
conditions which perturb the structure [22].

Fig. 2. Fourier-transform infrared (FTIR) spectra of rHA in the
amide III region for aqueous solution, pH 7.3 (a), and powder
lyophilized from pH 7.3 (b). The solid curves represent the
superimposed original spectra and the Gaussian fit, while the dashed
curves are individual Gaussian bands.

The foregoing example of albumin illustrates that an exchange between
thiols and disulfides is a prominent deterioration pathway for proteins
which contain these two moieties. Alternatively, a protein may contain
an even number of cysteines which are all disulfide-bonded to form
cystines (no free thiols). Such an example is provided by the classical
therapeutic protein insulin, a therapeutically important hormone which
is a strong candidate for use in a sustained-release device [23]. The native insulin molecule consists of two
polypeptide chains, with one intrachain and two interchain disulfide
bonds [24].

We have examined the stability of the insulin molecule (both bovine and
human forms) for the lyophilized powder stored at elevated temperatures
and humidities [25]. Figure 1b
depicts the time course of aggregation for lyophilized human insulin
stored at 50°C and 96% relative humidity. Closer examination of
the moisture-induced insulin aggregates indicated that both covalent
and non-covalent pathways could be responsible, depending on the
conditions of preparation and storage of the hormone [25].

Noncovalent aggregation of insulin can occur in, and has been studied
for, the aqueous solution where the deterioration is facilitated by
unfolding of the protein molecule at a hydrophobic (e.g., air--liquid)
interface [26]. The covalent aggregates were the
result of intermolecular disulfide bonding, as revealed by dissolution
in the presence of DTE [25]. The latter pathway
dominates for protein lyophilized from neutral to alkaline pH. It has
been shown independently that the insulin molecule is prone to
lyophilization-induced structural alteration, namely a 20% increase in
beta-sheet structure at the expense of alpha-helices and
unordered motifs [8]. It is unknown whether
alteration of insulin plays a role in its solid-state deterioration.

The finding that lyophilized insulin forms intermolecular disulfides was
puzzling considering that their formation requires the existence of
free thiols. The hypothesis was put forth [25]
that beta-elimination of the disulfides in native had occurred,
resulting in the formation of free thiols which could subsequently
catalyze disulfide exchange. In the beta-elimination step, a
hydroxyl ion facilitates the asymmetric cleavage of an intact S--S
bond, yielding dehydroalanine and thiocysteine residues. Both of these
are relatively unstable; the former can react with lysine to form a
lysin-alanine cross-link, and the latter decomposes to various thiol
products (e.g., HS-). The presence of such free thiols as a
result of the high temperature and/or high humidity incubation of
lyophilized insulin was directly confirmed experimentally [25].

Aggregation of lyophilized insulin was found to depend on the moisture
level. Incubation of bovine insulin at 50°C and various humidities
demonstrated that the extent of aggregation was greatly accelerated
above 65% relative humidity [25]. Concomitantly,
the water sorption by the lyophilized protein powder also began to rise
at this point in the adsorption isotherm. These data illustrate the
destabilizing effect of water on insulin, as was the case for albumin
(discussed above). In the case of insulin, water not only increases the
flexibility of the protein, thereby facilitating its aggregation, but
it is also a direct participant, in the form of hydroxyl ion, involved
in the beta-elimination step.

A special class of proteins are those created when a naturally occurring
macromolecule is covalently modified with formaldehyde to yield the
formalinized species. Such is the case of a deadly neurotoxin, tetanus
toxin, when it is detoxified by formaldehyde (diluted formalin) to
create the vaccine for tetanus, tetanus toxoid (TT) [27]. The reactivity of proteins with formaldehyde is
well-documented involving both stable and labile covalent formaldehyde
linkages [28-30], which occur
primarily with lysine, tyrosine, histidine, and cysteine residues [31]. Current efforts on developing a single-shot
vaccine for tetanus have renewed the interest in the stability of this
protein class [32].

As with insulin and albumin, we have observed that TT becomes insoluble
when the lyophilized protein is exposed to moisture [12, 32-34].
In Fig. 1c, the rapid time course of aggregation of
TT is seen under conditions of 86% relative humidity and 37°C.
Unlike previous studies with unmodified proteins in our laboratory,
insoluble aggregates formed under these conditions were held together
by covalent non-disulfide bonds, as indicated by their insolubility in
solvents containing 6 M urea and 10 mM DTE [32].
Acid hydrolysis of these aggregates revealed changes in the content of
strongly formaldehyde-binding amino acid residues--Lys, Tyr, and
His--implicating formaldehyde as the causal agent of the aggregation
[32].

By considering the known chemistry of formaldehyde reactions with
proteins, we devised a general mechanism to explain TT's aggregation
behavior. The proposed mechanism is based on the premise that
formaldehyde molecules are stored in the protein molecule as unstable
linkages, such as hydroxymethylamine. Once water is removed, reactive
Schiff base intermediates are formed that can combine with nucleophiles
from a second protein molecule to form either stable or unstable
covalent cross-links [32]. This mechanism was
supported by inhibiting aggregation by either succinylating TT or
reducing labile formaldehyde linkages with cyanoborohydride before
lyophilization [32]. Furthermore, aggregation
kinetics of another, unrelated, formalinized protein, diphtheria toxoid
(DT), was found to be superimposable with those of TT when lyophilized
from the same buffer solution [34]. Inhibition of
the aggregation of both DT and TT by succinylation supports the view
that the formaldehyde-mediated pathway is involved in both
instances.

An additional intriguing finding was the nearly complete inhibition of
aggregation of TT when co-lyophilized with small amounts of sorbitol
[12, 32]. The stabilization
effect could not be explained by structural or water content arguments
because sorbitol does not change appreciably the water content in the
protein powder, nor does it prevent the lyophilization-induced
structural alteration appearing in the solid state as determined by
FTIR [12]. However, when considering the
formaldehyde-mediated pathway, the possibility of the plentiful
hydroxyl groups of sorbitol out-competing the water molecules and
neighboring protein molecules for the reactive Schiff base appears to
be a plausible explanation for the stabilization effect [12].

OTHER PATHWAYS FOR DETERIORATION OF LYOPHILIZED PROTEINS

In addition to those described above in our case studies, other pathways
for protein deterioration may take place [1-4, 6]. Some of them are
intermolecular, e.g., aggregation, and others are intramolecular. It
has been hypothesized that intermolecular pathways are dominant due to
the close mutual proximity of protein molecules in the solid state [35].

Another potential pathway for protein cross-linking in the solid state
is the reaction between asparagine or glutamine and lysine, as
hypothesized for the moisture-induced aggregation of lyophilized
ribonuclease A [36] and recombinant bovine
somatotropin (rbSt) [35]. The latter protein also
undergoes noncovalent aggregation upon heating of its lyophilized
powder [37]. Increasing the moisture level
exacerbates the aggregation of both ribonuclease A [38] and rbSt [35, 37]. Enzymes suspended in organic solvents upon
heating also undergo a similar type of deterioration [39]. Furthermore, a mechanistic study [40] demonstrated the dimerization of solid insulin
(lyophilized from pH 4) involving the C-terminal asparagine of the A
chain and the N-terminal amino group of the A or B chain of another
molecule via a cyclic anhydride intermediate.

In addition to intermolecular pathways, intramolecular pathways may also
be responsible for protein deterioration in the solid state. These
mechanisms include: 1) deamidation of asparagine and glutamine
residues, particularly the former adjacent to a glycine residue; 2)
hydrolysis of peptide bonds, e.g., cleavage at the C- or N-terminal of
Asp residues; and 3) oxidation, especially of methionine residues. For
a more complete description of these and other mechanisms for protein
deterioration, including examples for lyophilized peptides and proteins
refer to [6].

RATIONAL STRATEGIES FOR STABILIZATION OF LYOPHILIZED
PROTEINS

Understanding the mechanisms responsible for deterioration of proteins
in the lyophilized state is more than merely a challenging intellectual
exercise, for obtaining such information is essential in rational
formulation development. The table lists examples of deterioration
pathways and of mechanism-based, rational approaches to
stabilization.

For proteins which undergo thiol-disulfide interchange, several
prevention strategies can be used. One simple approach is to lower the
pH of the aqueous solution prior to lyophilization to ensure the
protonated state of the thiol group of cysteine [13, 18]. The rationale for this
approach is that the ionogenic groups in proteins tend to retain their
ionization state upon lyophilization, as reported in a recent study of
model compounds [41]. Indeed, lyophilization of
BSA [13] or rHA [20] from
acidic solutions completely stabilizes the proteins against solid-state
aggregation during the high temperate and high humidity storage.

Another rational way to stabilize proteins against thiol-disulfide
exchange is to chemically block the thiol group(s) involved in the
process. For example, S-alkylating the Cys-34 of albumin stabilizes the
protein not only during high temperature and high humidity storage [13, 42], but also when loaded
within a polymeric matrix such as poly(fatty acid dimer:sebacic acid)
[42] and poly(lactide-co-glycolide) [43].

In the case of insulin, which is liable to beta-elimination
followed by thiol-catalyzed disulfide interchange, several
stabilization strategies were proposed and verified [25]. These include lowering the pH prior to
lyophilization (to both reduce beta-elimination and protonated
free thiols) and addition of Cu2+ to the formulation to
catalyze the oxidation of free thiols.

For formalinized proteins, whose moisture-induced aggregation involves
lysine residues, a useful approach is succinylation of the latter. The
generality of this approach was verified with both tetanus and
diphtheria toxoids [32, 34].

For any deleterious process involving intermolecular protein--protein
interactions, dilution of the solid protein should be effective. This
can be achieved by lyophilizing the protein from an aqueous solution
containing an inert polymer [13], e.g.,
poly(ethylene glycol). Also, since increased conformational mobility is
usually conducive to faster deleterious reactions in solid proteins,
and this mobility is enhanced by hydration, controlling the
latters extent by optimizing the humidity and moisture content in
the system is yet another powerful tool for stabilizing solid
proteins.

CONCLUDING REMARKS

In summary, studies on lyophilized pharmaceutical proteins demonstrate
their tendency to deteriorate, particularly via aggregation in the
presence of moisture. Thus, while it is true that lyophilized proteins
are usually more stable than those dissolved in water, this extra
stability is not unlimited.

Numerous deleterious mechanisms occurring in proteins have been
elucidated by us and others. The examples above illustrate how a
mechanistic approach is useful in solid protein formulation
development. This approach is fundamentally distinct from a simple
screening for a combination of operational and formulation conditions
which provide suitable stability. Some rules-of-thumb have been
proposed, as considered elsewhere [3-5, 44]. It is unlikely that a
universal strategy will be developed to formulate solid proteins, but
it is possible to develop some general strategies that are likely to be
effective against a variety of protein deterioration pathways. These
include the use of cryoprotectors (to protect against freezing) and
lyoprotectors (to protect against drying) which provide an amorphous,
glassy matrix for the protein where chemical reactivity can be kept to
a minimum. As additional, specific pathways for solid-state protein
deterioration are uncovered, it will become possible to develop
further, rational approaches to ensure high stability.

Although a measure of success has been achieved towards understanding
protein stability in the solid, e.g., lyophilized state, a number of
important issues remain to be fully understood. For example, the
question has not yet been definitively answered whether protein
structure, or rather the perturbation of protein structure in the dried
state, is necessarily related to the stability. Another significant
area is that of sustained delivery from polymeric matrices, and
assessing the utility of stabilization strategies (developed for
unincorporated protein) for the case of the protein formulation in
vivo. Such key areas are the focus of current and future research
towards understanding the deterioration of lyophilized proteins.
Moreover, with the advent of gene therapy, the problem of viral
instability, particularly acute for retroviruses, emerges as one of the
major bottlenecks [45]. For example, retroviruses
lose their activity during freezing, lyophilization, storage,
ultracentrifugation, and transfection. It remains to be seen to what
extent the protein stability/stabilization issues and strategies are
applicable to more complex, nucleic-acid-containing macromolecular
assemblies such as viruses.

This project has been financially supported by the National Institutes
of Health (grant GM26698) and the Biotechnology Process Engineering
Center at M.I.T.